Radioimmunoimaging Of Cetuximab Targeting EGFR In Basal-Like Breast Cancer

Objective:This study aimed to evaluate the feasibility of radioimmunoimaging with 99mTc and 68Ga-labeled the antigen-binding fragment(Fab)of Cetuximab for basal-like breast cancer.Methods:Fab fragment was obtained by papain digesting the monoclonal Cetuximab,sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE)was performed to detect molecular weight,and the specificity and affinity were detected by immunofluorescence staining and flow cytometry.Fab fragment was labeled with 99mTc by SHNH,68Ga by L-NETA and "click chemistry reaction between TCO and Tz.In order to evaluate the specificity of binding to EGFR,in vitro cell binding assays,SPECT imaging,small-animal PET imaging and biodistribution studies were carried out in EGFR positive basal-like breast cancer MDA-MB-468.Results:Fab fragment of about 50 kDa was obtained after digesting with papain,which could bind to MDA-MB-468 cells specifically in vitro,and the affinity did not change significantly compared with monoclonal antibody Cetuximab.99mTc-HYNIC-Cetuximab-Fab was synthetized with radiolabeling yield of 64%-87%and radiochemical purity of 92.37±4.88%,While 68Ga-L-NETA-Cetuximab-Fab was synthetized with high radiolabeling yield(about 80%)and radiochemical purity(>99%).Both of them can be taken up by MDA-MB-468 cells specifically in vitro with the highest uptake of 8.71±1.04%(99mTc,6h)and 12.35±0.19%(68Ga,2h).The results of SPECT imaging,small-animal PET imaging and biodistribution showed that MDA-MB-468 tumor had high uptake with 6.46±0.5%ID/g(6h)and 5.07±0.2%ID/g(2h)respectively.Moreover,the high uptake in tumor can be inhibited by excess Cetuximab(2.71±0.03%ID/g,2.25±0.16%ID/g,P<0.01).In addition,the liver and kidney had high uptake of the two probes,while the uptake of liver gradually decreases.Conclusion:99mTc and 68Ga-labeled EGFR antibody Fab fragment can bind to EGFR in vitro and in vivo with high specifically and affinity.This suggests it can be a radioimmunological agent for basal-like breast cancer detection.Objective: Among females,breast cancer is the most commonly diagnosed and the leading cause of cancer death,which seriously threatens females` physical and mental health.Basal-like breast cancer(BLBC)has special biological behavior and poor prognosis,while high expression of EGFR plays an important role in the development of most BLBC.Radioimmunoimaging(RII)is one of the important used tumor imaging techniques for nuclear medicine imaging.However,because of its large molecular weight and long circulation time,monoclonal antibody is not suitable for radionuclides with short half-lives(such as 18 F,68Ga,etc.).In pretargeting,the antibody is injected into the body first,after a suitable period of time,the antibody has accumulated to the tumor,and cleared from the blood,then injects a short half-life nuclide-labeled small molecule compound to radiolabel the antibody in vivo.Pretargeting can increase the concentration of the radiopharmaceutical at the tumor site,significantly increasing the tumor/background ratio.The purpose of this study was to explore the specificity of Cetuximab targeting EGFR and its value in PET imaging of basal-like breast cancer using pretargeting.Methods: Cetuximab-TCO was obtained by modifying Cetuximab with TCO-NHS.The radioligand 68Ga-L-NETA-Tz was prepared by using L-NETA as a chelating agent,the labeling rate and in vitro stability were determined by radio-HPLC.Human basal breast cancer cells MDA-MB-468 and MDA-MB-231 were cultured in vitro,and EGFR expression level of the two cells was identified by western blot.In vitro experiments explored the specificity of the probe and the feasibility of pretargeting.Nude mice bearing xenografts of the above two cell lines were established.50?g of Cetuximab-TCO was injected into the tumor-bearing mice in advance,and 150?Ci 68Ga-L-NETA-Tz was injected at different times(48,36,24 and 12h),and pretargeting was realized through “click chemistry”.Small-animal PET imaging and biodistribution were performed to evaluate pharmacokinetic properties and specificity of the probe.Results: The 68Ga-L-NETA-Tz molecular probe was successfully prepared with a labeling yield of >95%.After the probe was placed in fetal bovine serum for 2 h at 37?,the radiochemical purity was >95%.Western blot and immunohistochemistry showed that the expression level of EGFR in MDA-MB-468 was higher than that in MDA-MB-231.In vitro cell uptake experiments showed that although antibody internalization gradually increased,there were still some binding sites on the cell surface,confirming the feasibility of the pretargeting.PET imaging and biodistribution results showed that the best imaging results were obtained at 36 h pre-injection,achieving the highest tumor uptake(0.77 ± 0.05% ID/g,1h),and optimal tumor/muscle ratio(4.67 ± 0.46).There was no significant uptake of tumors in the blocking group,the control group without injecting Cetuximab-TCO,and the MDA-MB-231 negative control group.Conclusion: In this study,we successfully enable the radioimmunoimaging of monoclonal antibody radiolabeled with short half-life nuclide.The pretargeting with Cetuximab-TCO and 68Ga-L-NETA-Tz could detect the expression of EGFR in vivo and provide a novel method for PET imaging of basal-like breast cancer.